Biochemical and Biophysical Research Communications
Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region
Highlights
► We determined the crystal structure of human FAF1 UBX domain. ► The structure reveals a novel FcisP touch-turn motif in p97-binding region. ► The structure presents two different conformations of p97-binding region. ► The two conformations should represent the unbound and bound states.
Introduction
p97/VCP, a multi-functional ATPase of the AAA+ family, is implicated in diverse cellular processes such as post-mitotic Golgi reassembly, endoplasmic reticulum associated degradation, nuclear envelope reconstruction, cell cycle, suppression of apoptosis and DNA damage response [1]. The diverse functions of p97/VCP are mediated by the interaction with various adaptor proteins in each different biological context [2], [3]. The largest group of the adaptors comprises the proteins containing the UBX domain, such as FAF1, p47, SAKS1, and UBXD7, which directly binds to N domain of p97/VCP [4], [5], [6], [7]. The UBX domain comprises about 80 amino acid residues and is currently considered as a general p97/VCP-binding module, with a growing number of UBX proteins being identified [8]. The structures of UBX domain were determined by NMR spectroscopy on FAF1 UBX and p47 UBX, revealing that it adopts a β-grasp fold similar to ubiquitin [5], [9]. The first structural glimpse on its interaction with p97/VCP was obtained from the crystal structure of p47 UBX in complex with p97/VCP ND1 fragment at 2.9 Å resolution [10]. It showed that the conserved FP sequence motif in the loop connecting strands S3 and S4 is inserted into the cleft of the p97/VCP N domain and is essential for the binding activity [10].
FAF1 was initially identified as a Fas-interacting protein in the death inducing signaling complex and was shown to potentiate the apoptotic cell death [11], [12], [13]. Later, FAF1 was shown to intervene in the NF-κB signaling at two points of the pathway. First, FAF1 binds to the IKK β subunit, thereby disrupting the IKK complex assembly and inhibiting its activation [14]. Second, FAF1 also binds to p65 subunit of NF-κB and inhibits the translocation of NF-κB into the nucleus [15]. Most recently, FAF1 was revealed to inhibit the proteasome-mediated protein degradation process either by interacting with p97/VCP which may serve as a molecular chaperone presenting the ubiquitinated client proteins to the proteasome or by interacting with ubiquitinated client proteins [4]. As a consequence of its involvement in these cellular processes, FAF1 acts as a pro-apoptotic protein.
Human FAF1, composed of 650 amino acid residues, contains a UBX domain at the C-terminus through which it binds to p97/VCP N domain [4], [16]. In an effort to elucidate the conserved structural features of the UBX domain for its binding to p97/VCP and also to provide a structural basis of the interaction between FAF1 and p97/VCP, we initiated X-ray crystallographic analysis on FAF1 UBX domain, and here we present its crystal structure determined at 2.9 Å resolution.
Section snippets
Protein preparation, crystallization and data collection
The human FAF1 UBX domain (residues 571–650) was overexpressed, purified and crystallized as described previously [17]. Briefly, FAF1 UBX protein was overexpressed in fusion with His-tagged thioredoxin in Escherichia coli strain Rosseta2 (DE3). The fusion part was removed by TEV protease during the purification process. The FAF1 UBX alone was concentrated to 9.7 mg/ml for its crystallization. We collected X-ray diffraction data to 2.9 Å resolution, at Photon Factory, Japan on the beamlines
Overall structure of FAF1 UBX
FAF1 UBX domain comprises five β-strands (S1–S5), one α-helix (H1), and three 310 helices (G1–G3) (Fig. 1A). The five strands form a mixed β-sheet in the order of 2–1–5–3–4, and the sheet rolls around the α-helix H1 to form the well-known β-grasp fold, as previously shown in the NMR structure of FAF1 UBX [9]. Notably, among the three short 310 helices G1 to G3, only G3 was recognized in the previous NMR structure as a helix H2.
FAF1 UBX domain contains three Phe-Pro sequence stretches: FP608-9,
Acknowledgments
This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080930). The authors thank the staff of the Pohang Light Source beamlines 4A in Korea, and the Photon Factory beamlines AR-NW12 in Japan.
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